Method and apparatus for classifying and segregating particles with electrical and optical means

ABSTRACT

Apparatus for classifying, counting and segregating microscopic particles such as cells. The particles may be contained in a gas or liquid and may be classified by measuring two characteristics thereof such as the size of the nucleus and the size or volume of the cell. This permits to count and separate red blood corpuscules from leucocytes in the same solution. Normal and atypical cells may be physically separated. The size of the nucleus may be measured by transmitted light and the size of the cell by variations of an electromagnetic field or by scattered light.

United States Patent Legorreta-Sanchez [451 July 11, 1972 54] METHOD AND APPARATUS FOR 2,942,689 6/1960 Walker et a1 ..324/61 x CLASSIFYING AND SEGREGATING 3 223.43; 2/1962 PARTICLES WITH ELECTRICAL AND 3 1966 Coulter et al [72] inventor: Glldardo Legorreta-Slnehez, 2159 Mexico m PUBUCATIONS Avenue, Guadalajara Mexiw Clark; .l. w. and Randall; .l. e. An Electromagnetic Blood [22] Filed. Mmh 17, 1969 Flow Meter, in The Review of Scientific Instruments. Vol. 20,

' No. 12, Dec. 194 pp- 951- 954. 0184.118. [21] Appl- N -Z 809, Mattern; C. F. T., Brackett; F. S. and Olson; B. .I. Determination of Number and Size of Particles by Electrical Gating in APPMM Journal of Applied Physiology. vo. Jan. 1957. pp. 56- 7o. [63] Continuation of Ser. No. 435,377, Feb. 25, 1965,

abandoned. Primary Examiner-E. E. Kubasiewicz Attorney-Wham & McManigal [52] U.S.CI. ..209/4, 209/1l1.7,209/11l.8,

210/65, 324/71, 324/34, 324/61, 356/39 ABSTRACT [2;] Int. CL. Apparatus for classifying counting and segregating micm l l c 4 I 7 i 6 scopic particles such as cells. The particles may be contained in a gas or liquid and may be classified by measuring two characteristics thereof such as the size of the nucleus and the [56] CM size or volume of the cell. This permits to count and separate UNITED STATES PATENTS red blood corpuscules from leucocytes the same solution. Normal and atyplcal cells may be physlcally separated. The Berry size of he nucleus may be measured transmitted and 5 I/ 1960 PP" "235/92 the size of the cell by variations of an electromagnetic field or 3,233,173 2/1966 Lees et ....324/6l by scanned light 3,361,965 2/1968 Coulter et al. ....324/71 3,390,326 6/1968 Imadate ....324/61 Clahm, 7 Drawing Figures 4 [fay/' 2 [av/liar lmfI/ficr Amp/I flier 77 l l f I a 2 I f I 1%! 149? Pills: l/e/yl/ Pulse #4) H: flzy i/ D/Qsc. D in. fl/Jc. 0115c Z e9 j 7 4n// Ivy/r7 [a/nc l Jtnc e Anv l/fie! Circa/f (am/er l 12 i .55 l (3- eel- 5/ 56 36 3 7 ZJ 48 a 33 44 30 METHOD AND APPARATUS FOR CLASSIFYING AND SEGREGATING PARTICLES WITH ELECTRICAL AND OPTICAL MEANS This application is a continuation of my copending application Serial No. 435,377, filed Feb. 25, 1965, now abandoned.

This invention relates generally to the automatic investigation of microscopic particles, and more particularly relates to a method of and apparatus for classifying, counting or segregating particles such as cells suspended in a liquid in accordance with certain characteristics thereof.

Techniques of observing particles through a microscope are constantly being improved. These techniques include those utilizing monochromatic light for illuminating the object, such as ultraviolet light which, of course, improves the resolution. Recently, various techniques have been developed to obtain automatic observations by means of a microscope. This includes the use of electronic photomultipliers and other light sensitive devices to measure the change in light intensity or to detect the color of an object to be observed. They may also be used to measure the light received from individual spots of the object which form the image so that the light intensity or the area of an image may be recorded and measured. in order to obtain such automatic observations, various electronic devices have been attached to the microscope to process the electric signals, such as pulses, to which the image may be converted. In some cases it has even been suggested to utilize electronic memory devices and computers to store information and to process it. The development of techniques of processing biological tissues by histochemical methods even permit the identity of a specific substance within a cell.

Some of these recent procedures have found clinical application, others are used as research tools to provide information on the physiology of cells. The result of this continuous improvement in tissue processing and improvements in microscopes and electronics utilized in connection with a microscope to process information automatically are apparatuses such as the cytoanalizer, the flying-spot microscope, utilizing a flying-spot television technique, a television microscope where the image produced by the microscope is televised, the latter may even use ultraviolet light. Additionally, such devices include a microdensitometer, microcytophotometer, microspectrophotometer, or fluorometric devices.

However, regardless of the sophistication of the optical or electronic components, all of these devices have a common feature. The object to be investigated is deposited or attached to a transparent material such as a glass slide. The glass slide is put on the microscope stage and, accordingly, the object can be made to move roughly within the focal plane of the microscope. The focal plane of a microscope may be defined as an area between the focal length and twice the focal length of the microscope objective. It is known that all objects within that area produce a sharp image through the optical system.

The fact that the object must be attached to a transparent material or glass slide has a number of disadvantages. Thus, when the glass slides are changed to observe another object, the new object moves into the area of observation along the focal plane of the microscope. This is also true when the glass plate is moved to observe another object thereon. The result of this movement is that the new object is no longer in focus. This is primarily due to the fact that the thickness of the focal plane is very small so that any object readily moves out of the focal plane. For this reason, all microscopes have a coarse and a fine adjustment which cause movement of the objective. Hence, in order to produce a sharp image, it is almost always necessary to adjust at least the fine vertical movement of the microscope.

Some microscopes used for automatic work have a stage coupled to a scanning apparatus so that the stage is caused to have oscillatory movements such as a zig-zag movement. This makes it possible to observe the entire surface of the glass slide on which some object has been deposited. During this scanning operation the object frequently goes out of focus making it impossible to produce a sharp image. This, in turn,

leads to error and makes it impossible to continue the automatic scanning. Various attempts have been made in the past to overcome this drawback. However, this requires complicated arrangements and, therefore, none of these has succeeded.

Apparatus is available commercially at this time where blood cells are counted by diluting them in transparent liquid, and letting them move along the focal plane of a microscope or under dark field illumination. Hence, an object moving along the focal plane will scatter the light and produce pulses in a photomultiplier attached to the microscope. Hence, the individual cells move parallel to or within the focal plane of the optical system. If the particle moves at right angles to the focal plane within the capillary tube along which the object moves, the object, of course, goes out of focus. In addition, the curved capillary tube, the curved interface between the liquid and the surrounding air cause a very complex optical system which is not suitable for fine work due to refraction and scattering of the light.

It is, accordingly, an object of the present invention to provide a method of and apparatus for moving an object across the focal plane of an optical system in such a manner that the object is momentarily and precisely in the focal plane only once.

Accordingly, the main requirement of the method of the present invention is to suspend the object to be investigated in a fluid. Therefore, this method for the automatic observation of biological samples is limited to such material that naturally exists in the form of particles. Those are, for example, the cells of the blood, cells or small groups of cells spontaneously exfoliated from the epithelial surfaces and other body surfaces and those that are naturally or pathologically but spontaneously found in the urine, the peritoneal fluid, the pleural fluid, the vagina or the cervix, the respiratory tract, the alimentary tract, or the surface of the skin.

The present invention is particularly directed toward the classification, counting or physical segregation of biological cells. Cells usually have a nucleus containing desoxyribonucleic acid, a cytoplasm and an outer membrane surrounding the cytoplasm. The present invention is particularly related to the classification of those cells which cover the epithelial surfaces of the body. These surfaces continuously exfoliate either single cells or small, loose groups of cells.

The red blood corpuscles or erythrocytes and the white blood corpuscles or leucocytes are freely suspended in the plasm of the blood. Thus, these are cells normally suspended in a liquid. It should also be mentioned that normal red blood corpuscles, at least in the human being, do not have a nucleus, but the white blood cells do.

it is frequently necessary for diagnostic and other medical purposes to count the red and white blood cells of a patient. This is usually efi'ected by first diluting the blood one hundred times whereupon those red blood cells contained in a small chamber of known dimensions are counted under the microscope. The white blood cells are counted by first diluting the blood twenty times and by chemically destroying the red blood cells. Thereafter, those white blood cells contained in a similar counting chamber of known dimensions are also counted under the microscope.

[t is also frequently required to classify white blood cells according to their morphology. This may require the measure ment of the size of the nucleus as well as the size and staining reaction of the cytoplasm. This is presently effected by smearing a thin film of blood on a glass slide, staining it in a conventional manner, and to study under a microscope a certain number of white cells to classify them.

Attempts in the past to count both red and white blood cells simultaneously were abandoned. Thereason for this is that in normal persons out of 80,000 cells only about are white cells. Therefore, in order to obtain an accurate count of both red and white blood cells, it would be necessary to count is very large number of cells. Such a procedure is obviously very time consuming and expensive.

n the other hand, in order to count the red and white blood cells separately the blood must be diluted 100 times for counting the red cells and only times for counting the white cells. If any mistake is made in either one of the two dilutions, the figures obtained for the white and red blood cells are wrong which might lead to an erroneous diagnosis.

Recently, a machine has been introduced for automatically counting blood cells by means of an electrical impedance principle. An electric current is established through a conductive liquid having cells suspended therein. When a cell crosses the path of the electric current, the intensity of the current is momentarily decreased. The resulting electric pulse may then be amplified and counted to determine the number of cells. it should be noted, however, that this machine only permits the counting of the number of cells such as either red or white blood cells. Platelets are non-nucleated particles which normally exist in the blood in much smaller numbers than the red blood cells. They are counted together with the red blood cells and this error may be disregarded. However, this machine does not permit to measure any characteristic of the cells.

it has also been proposed to count cells by optical gating. Here, each cell moves in the focal plane of a microscope which may be arranged with a dark field illumination. Accordingly, the focal plane is dark until light is scattered by a particle such as a cell within the focal plane. This scattered light is made to impinge upon an electronic photomultiplier to produce an output pulse which may then be counted to determine the number of cells or particles.

However, with both of these known machines, the red and the white blood cells have to be counted separately and a different dilution of the blood must be made to permit the separate counting of the erythrocytes and leucocytes.

it is accordingly one of the objects of the present invention to provide a method of and apparatus for counting both red and white blood cells simultaneously by using the same dilution and obtaining both the number of the red and of the white blood cells.

Another object of the invention is to provide a method of and apparatus for classifying white blood cells or other cells in accordance with predetermined characteristics such as, for example, the size of the nucleus and the staining reaction and the size of the cytoplasm.

It has long been recognized that the study of cells exfoliated from the epithelial surface of a human being, for example, permits the diagnosis of particular diseases, including the occurrence of certain types of cancer. For example, cells exfoliated from the female genital tract, such as the cervix, the urinary tract, the respiratory tract and other epithelial surfaces may include both normal cells and atypical or abnormal cells. From the research of the last twenty years it is well known that an atypical cell may be differentiated from a normal cell by the size of its nucleus, the volume of the cell, the absorption of ultraviolet light, particularly by the nucleus, the absorption of light after the nucleus has been stained, the emission of light when the cell has been stained with fluorescent stains and illuminated with ultraviolet light or the relation between the size of the cytoplasm and the size of the nucleus.

These criteria form, in part, the basis of the exfoliative cytology method of diagnosing certain types of cancer. This is the well-known Papanicolaou smear method for diagnosing cancer of the cervix, lungs and the like.

According to the cytologic method of Papanicolaou cells exfoliated from an epithelial surface are smeared on a glass slide, fixed and stained for observation under the microscope. The method may include the filtration of large amounts of urine, pleural, or peritoneal effusions through a membrane filter that retains both normal and abnormal cells. Then, the filter is attached to a glass slide, stained and mounted for study under the microscope. This procedure, of course, permits the concentration of both normal and abnormal cells. However, the proportion of normal and abnormal cells remains the same.

A further improvement that may be mentioned here is the use of trypsine, hyalorunidsase, and other chemicals to dissolve mucous-like substances which may surround some exfoliated cells such as sputum. The cells are then suspended in a liquid and filtered in the manner above-described in connection with urine. Again, the proportion of normal and abnormal cells remains the same as that in the original sample.

The cytologic observation of exfoliated cells by the Papanicolaou method has proved to be very effective for the early detection of cancer. Some authors contribute to the application of this method the statistically observed decrease in deaths due to cancer of the cervix. Many medical authorities advise that every woman afier a certain age should have a genital cytological examination made at least once every year. Hence, this procedure is currently in use either for the diagnosis of patients having certain symptoms or in asymptomatic individuals to detect cancer in its earliest stages. Obviously, the earlier cancer is diagnosed, the better the chances of curing it.

Although the Papanicolaou method is of amazing accuracy, it still has some limitations. As indicated above, normal and atypical, or cancerous cells occur mixed with each other. Usually, the atypical or cancerous cells occur in very small proportion particularly during the early stages of the disease. Therefore, the slides on which the cells are mounted must be screened looking for atypical cells. It takes from live to twenty minutes to screen a single slide. So, in any busy laboratory, that is, in practically all of them, the screening is effected by a technician who marks those areas which require review by a cytologist. Therefore, a screening technician can usually examine from 40 to 60 cases a day. There are not enough such technicians available to perform all the necessary work. The time required for this job and the necessary skill of the technicians involved have the result that this routine investigation becomes quite expensive.

Nevertheless, a false negative report occurs in about ten per cent of the positive cases. To a certain degree, this is due to the fact that there are so few suspicious cells, particularly in the early stages of the disease. Accordingly, the suspicious cells may be overlooked. In addition, the cells must be obtained from a sample and only a small portion of the sample is smeared and examined. Where the sputum is examined, the portion actually investigated may be less than one hundredth of the whole sample. Unfortunately, therefore, a negative report does not exclude the possibility of cancer.

It is accordingly a further object of the present invention to provide a method of and apparatus for classifying automatically cells suspended in a liquid into those which are normal and those which are atypical and might be cancer cells.

Still another object of the present invention is to classify automatically cells into normal and atypical or cancerous cells suspended in a liquid and to physically segregate them from each other thereby to provide a high concentration of suspicious cells, that is, those which are either atypical or possibly cancerous.

it should be noted that the segregated cells are subsequently filtered through a membrane filter which retains all of the selected suspicious cells. The membrane filter with the cells retained on it is stained by the Papanicolaou method and diagnosed under the microscope in the conventional cytologic procedure.

it will be apparent that a segregation of normal and atypical cells should prove of great advantage to the medical field and should permit a more accurate diagnosis with a smaller expenditure of time and effort. Thus, where the suspicious cells, that is, atypical or even cancerous cells are highly concentrated, the diagnosis by the classical methods of cytology is obviously more accurate and can be rapidly performed by a trained cytologist.

it is also frequently desired to study the function of the ovary. To this end, it is necessary to count and to classifyunder the microscope several hundred cells exfoliated from the vaginal epithelium studying their size and the size of the clumps of cells. Such a procedure permits a study of the ovary as well as the effects of hormones which might have been administered. For practical reasons, it is not possible to count and classify more than say 300 cells. On the other hand, since the number of cells involved in the count is rather small the result may be inaccurate.

It is accordingly still a further object of the present invention to count and classify cells or clumps of cells exfoliated from the vagina by classifying them in accordance with the size of the cytoplasm and the size of the nucleus.

It should be emphasized that the apparatus of the invention only classifies cells in accordance with predetermined criteria and may automatically segregate them in accordance therewith. However, any diagnosis on the basis of the cells still must be made by a highly skilled person such as a cytologist. The apparatus simply increases the number of suspicious cells compared with the number of normal cells.

It should also be noted that quanitative measurements of cell properties have previously been performed to serve as a basis for screening cells into normal and atypical cells. This has been reported by W. J. l-lorvath, W. E. Tolles, and R. C. Bostrom in the I956 Transactions of the First International Cancer Cytology Congress, page 371. For example, FIGS. 4 and S of this paper compare normal cells with those from cancer patients. For each cell, the ratio of cell diameter to nucleus diameter is measured as well as the nucleus extinction coefficient. The two figures clearly show that for cancerous cells, the ratio of cell diameter to nucleus diameter has decreased considerably indicating an increase of the diameter of the nucleus for cancerous cells as well as a smaller cytoplasm. A large nucleus and a proportionally small cytoplasm characterize neoplastic cells.

FIG. 6 of the same paper is a graph showing the per cent of cells exceeding certain criteria which coincide with confirmed cancer. Thus, this makes it clear that normal cells may be segregated from suspicious or atypical cells. In accordance with the present invention, this classification and physical segregation of the cells is done automatically and in a liquid medium.

Sometimes it is desirable to classify, count or segregate for further study particles suspended in a. gas such as air. These particles may be liquid, solid, or of biologic nature. For example, this may be necessary to study air pollution, the dust levels in mines, factories or mills, or in research clean rooms. It may also be desirable to study and monitor the size distribution of aerosols or powdered industrial products.

It is accordingly a still further object of the present invention to provide an apparatus for and method of counting, classifying or segregating particles suspended in a gas.

In accordance with the present invention the cell size is measured. To this end, the particles or cells are suspended in a liquid and the suspended particles are caused to flow past a predetermined testing volume. A steady electromagnetic field is established within this testing volume. The change of the electromagnetic field caused by the passage of a particle or cell through the testing volume is measured. This, of course, determines the size of the cell.

The size of the nucleus is preferably measured by means of light. Thus, the nucleus may be stained to make it absorb visible light or fluorescent stains may be used to emit light when irradiated or else ultraviolet light may be used which is absorbed by the nucleus. Thus, light may be projected through the particle. Preferably, a compound microscope is utilized. A photomultiplier may be attached to the ocular of the microscope. This will detect and amplify the change in the light output caused by a nucleus which absorbs light. This permits to determine the size of the nucleus. This, in turn, makes it possible to determine the ratio of the sizes of the cytoplasm and the nucleus. Any cell having a particular size of the cytoplasm and of the nucleus or preferably a particular ratio may be separately counted and may be segregated from the other cells for further inspection by a cytologist.

The novel features that are considered characteristic of this invention are set forth with particularity in the appended claims. The invention itself, however, both as to its organization and method of operation, as well as additional objects and advantages thereof, will best be understood from the following description when read in connection with the accompanying drawings, in which:

FIG. 1 is a schematic view and block diagram of an apparatus embodying the present invention for investigating and segregating cells in accordance with their characteristics such as the size of the cytoplasm and of the nucleus;

FIG. 2 is an isometric view of the diaphragm including the focal plane of the compound microscope of FIG. 1, and illustrating two electromagnets for establishing a steady electromagnetic field and means for adjusting the size of the effective aperture;

FIG. 3 is a vertical sectional view through the area about the focal plane of the microscope, and illustrating particularly the light condenser, the objective and the means for adjusting the effective aperture;

FIG. 4 is a schematic vertical sectional view of a modified microscope utilizing ultraviolet light which may be used in accordance with the present invention;

FIG. 5 is a schematic sectional view of a modified apparatus for segregating cells including an electric capacitor for developing a steady electrostatic field;

FIG. 6 is a schematic sectional view of a device similar to that of FIG. 5 but utilin'ng electrodes for establishing an elec tric fieid; and

FIG. 7 is a schematic sectional view of a light condenser which may be used in connection with a microscope shown particularly in FIGS. 1 and 3.

Referring now to the drawings and particularly to FIGS. 1 through 3, there is illustrated an apparatus in accordance with the present invention for investigating microscopic particles such as cells and for segregating them in accordance with certain predetermined characteristics.

As shown particularly in FIG. 1, the apparatus includes a conventional compound microscope indicated generally at I0. The light source 11 is associated with the microscope l0 and may be a lamp having a ribbon filament and a pair of leads l2 connected to a suitable power supply and a suitable lens 13 for directing parallel light as indicated by the arrow 14 toward the microscope 10. The microscope includes a conventional light condenser 15 which may be a lens as shown for focusing the light onto the focal plane of the microscope. The microscope further conventionally includes an objective [6 which may be a compound lens as shown and may include three oculars or eye pieces 17. A system of light-splitting mirrors 9 or a conventional prism system may be used to split the single light beam from the objective 16 into three light beams which are received respectively by the eye pieces 17. It should be emphasized that while the compound microscope I0 is conventional, it does not require a revolving turret with a plurality of objectives to change the magnification. Also, the microscope 10 does not require any means for changing the focus nor does it require the conventional stage.

Disposed above each ocular 17 is a light-sensitive device such as photomultiplier I8 including a photosensitive cathode and a plurality of dynodes for multiplying the electron steam resulting from light impinging on the cathode. Between each ocular 17 and its photomultiplier I8 a diaphragm 19 may be disposed that masks a portion of the image such as a third of the image. However, each diaphragm 19 should mask a different portion of the image of the focal plane of the microscope. Accordingly, the photomultipliers l8 jointly see a complete image of an object in the focal plane. The photomultiplier l8 conventionally includes a well-regulated power supply. It is well known that the amplification obtained by a photomultiplier is critically dependent on the accelerating voltages applied to its dynodes. On the other hand, adjustment of the supply voltage permits ready control of the amplification of the photomultiplier. Instead of using a photomultiplier other conventional devices may be substituted such as a photocell or a TV camera without departing from the spirit and scope of the present invention.

The electronic circuitry illustrated schematically in block form in FIG. 1 will be subsequently explained.

There is provided a container 20 which contains a suspension of the microscopic particles to be classified and sorted. The container 20 may be provided with a scale 21 for measuring the volume of the liquid within the container. Thus, the container 20 may contain a suspension of cells. Epithelial cells to be studied may be processed in a conventional manner as follows:

The sample of the exfoliated cells to be classified and sorted is mixed with a physiological saline solution which is then shaken. Some samples require chemical digestion of the mucous-like substance surrounding and holding together the cells by one of several known methods. The solution containing the cells is filtered through a very fine sieve to remove big clumps of cells. Then, the solution is put into a centrifuge to concentrate the cells which form a sediment. This sediment is chemically stained in any conventional manner in order to stain only the nucleus of each cell. Subsequently, the sediment is washed with a saline solution. This solution is again subjected to centrifugation to eliminate the remaining chemical stain. Then, the cells are suspended again in a saline solution. In the case of blood, the blood may be diluted in a liquid including a suitable stain for staining the white blood cells. As a result of this treatment, the nucleus is stained while the outer cytoplasm remains practically without color. After the cells have been segregated, the suspension of the cells is filtered through a membrane filter to retain the cells. The filter is mounted on a glass slide and studied under the microscope. This procedure is conventional for cytologic investigations.

However, it should be noted that staining of the nucleus is not always required. All that is needed is to utilize a light or such a color or wave length including ultraviolet light that it is absorbed by the nucleus. [t is well known that the nucleus of a cell consists essentially of desoxyribonucleic acid which absorbs ultraviolet light of the order of 2,500 AU. Thus, by using ultraviolet light of this wave length range and a suitable microscope having a quartz or a fluorite optical system, the nucleus will absorb the ultraviolet light without the necessity of staining it.

Alternatively, after staining the cells it is also feasible to utilize suitable color filters for differentiating between the color of the nucleus and the cytoplasm or for distinguishing other properties of the cells. This may make it possible, for example, to distinguish the age of the cells or to some extent their place of origin. in this connection, it is also feasible to utilize a microdensimeter and a fluorometric device for determining color absorption. ln this manner leucocytes are classified.

A closed chamber 22 surrounds the light condenser and the microscope objective l6 and is connected by a suitable conduit 23 to the container 20, preferably, by means of a valve 24. It should be noted that the volume generally indicated at 25 between the condenser 15 and the objective 16 and including the focal plane of the microscope may be filled with the suspension of cells from the container 20. Accordingly, the outer lens of the objective l6 and the outer lens of the condenser 15 are in contact with the liquid. A flask 26 is provided with an air-tight stopper 27 through which extends a hollow tube 28. The tube 28 may be connected by a plastic and nondeformable hose 30 to another tube 31 extending into the lower portion of the container 22 surrounding the microscope objective and condenser.

By means of a conduit 32 connected to the neck of the flask 26, the flask is connected to a vacuum system schematically indicated at 33. Accordingly, when the valve 24 is opened conduit 23, container 22, and conduits 30 and 32 are put under reduced pressure to aspirate the suspension in the container into the container 22 and eventually into the flask 26.

Similarly, flasks 34 and 35 are each connected by respective conduits 36 and 37 to the container 22. This is effected by means of tubes 38 and 40 each of which terminates very closely to the focal plane of the microscope in. the area between the condenser l5 and the objective 16. In turn, each of the flasks 34 and 35 is connected to the vacuum system 33 by conduits 41 and 42. A valve 43 interconnects the conduit 36 to the flask 34. Similarly, a valve 44 is connected between the conduit 37 and the flask 35. This makes it possible to selectively connect either the flask 34 or 35 to the vacuum system 33 thereby to suck any microscopical particles such as a cell by the action of the vacuum into the selected flask.

As shown particularly in FIGS. 2 and 3, there is provided a diaphragm or dividing wall 46 which separates the container 22 into an upper chamber 47 and a lower chamber 48. It will be noted that the exhaust tubes 3] 38, and 40 for the cells are disposed in the lower chamber 48. This diaphragm 46 is electrically insulating and opaque to whatever light is used in the optical system. This diaphragm 46 is provided with a small central aperture 50 through which the light is projected by the condenser 15 and across which the focal plane of the microscope extends.

The size of the opening 50 is made adjustable by means of two movable knife edges 51. These are adjustable by the knurled nuts 52 rotatable but not slidable in a slot in a housing extension 49 and disposed about the screwthreaded end 53 of each of a pair of bars 54 which, inturn, carry or are integral with the knife edges 51. Thus, by rotating the knurled nuts 52 in one direction or the other, the two knife edges 51 can be made to approach each other or to recede from each other thereby to adjust the size of the aperture through which the light is permitted to pass into the microscope. Of course, the knife edges 51 are equally opaque and nonconductive.

In accordance with the present invention, means are also provided for generating a steady magnetic field between the upper chamber 47 and the lower chamber 48. instead of a magnetic field, it is also feasible to provide an electric field, the term electromagnetic field being used to cover both. For developing a magnetic field, there are provided two opposed iron rods 55 (see FIGS. 1 and 2) extending through the diaphragm 46 into the neighborhood of the central opening 50. A coil 56 is disposed at the outer end of each of the iron rods 55. Each of the coils 56 is connected to a suitable electrical power source such as battery 61 by means of leads 57. Hence, the electric current flowing through the two coils 56 will generate a steady magnetic field which exists particularly in the neighborhood of the focal plane of the microscope. Alternatively, a steady electric field may be developed by electrodes & will be described hereinafter in connection with FIG. 6 or by using a capacitor as illustrated in FIG. 5. Again, the electric field should be provided in the neighborhood of the focal plane.

Finally, a pair of electrical pickup electrodes is preferably provided in the neighborhood of the diaphragm opening 50. To this end, there may be provided a metal ring 58 which may, for example, be disposed about the condenser lens 15. Similarly, another metal ring 60 may be disposed about the objective 16 of the microscope.

The operation of the apparatus illustrated in FIGS. 1 to 3 will now be explained.

At first, a suspension of the cells is produced in the manner previously described. The nucleus may be stained or else light may be used of such a wave length that it is absorbed by the nucleus of the cells in question. ln any case, the suspension is put into the container 20, the valve 24 is opened, and the vacuum system 33 is started so that both the upper chamber 47 and the lower chamber 48 are filled with the suspension.

It may be advisable to provide the container 20 with a conventional magnetic stirrer. This includes a rotating magnet which, in turn, causes the rotation of a suitable stirring mechanism. Otherwise, the cells in the suspension may tend to settle down.

Then the light source 1] is turned on and the photomultiplier is supplied with power. Also, the two coils 56 are connected to their battery 61. The apparatus is now ready to be used.

The liquid used for suspending the cells is preferably but not necessarily electrically conductive. However, it preferably has a high electrical resistance. in any case, the dielectric constant of the liquid should differ from that of the cells to be classified.

By virtue of the study magnetic field which is generated by the rods 55 and the coils 56, the passage of any cell through the central aperture 50 may be readily determined. The passage of a cell will disturb the magnetic field. The size of the disturbance will indicate the size of the cell. This change of the magnetic field can be sensed by the two electrodes 58 and 60. The signal picked up by the electrodes 58, 60 is an electric pulse which may be amplified by an amplifier 65.

To this end, the amplifier 65 has two input leads 66 and 67 connected respectively to the electrodes 60 and 58. The output of the amplifier 65 may be fed to two pulse height discriminators 70 and 71 connected in parallel. Each of these discriminators is arranged as a conventional differential pulse height discriminator so that it will transmit only electrical pulses having an amplitude or pulse height between two predetermined values.

The output of the pulse height discriminator 70 is fed to an amplifier 72 which, in turn, controls the valve 44 which is preferably a solenoid valve. Similarly, the output of pulse height discriminator 71 is fed to amplifier 73 which controls the solenoid valve 43. Thus, the valves 43 and 44 are opened by their respective amplifiers 72 or 73 which, in turn, are controlled by the pulse height discriminator 70 and 71.

To summarize, therefore, the valves 43 and 44 are opened in response to a cell passing through the aperture 50 which has a volume or diameter between two predetermined limits. These limits are adjustable by adjusting the discriminators 70 and 7]. Depending on the size of the cell, it is either sucked in by the conduit 38 or by the conduit 40 and eventually deposited into either flask 34 or 35. On the other hand, if the volume of the cell is outside of the predetermined limits, the cell is discarded and eventually collected in the flask 26. Those cells are the normal cells which are of no further interest.

Further, in accordance with the present invention, the cells may also be classified and eventually segregated in ac cordance with the size of the nucleus. This is effected by the compound microscope 10. Every time a cell passes through the opening 50 from above to below, the focal plane of the microscope, one of the photomultiplier tubes 18 receives less light because the nucleus absorbs some of the light. This decrease of light causes a decrease of the electrical current at the output of the photomultiplier. The outputs of the photomultipliers 18 are gated through an anticoincidence circuit 69 having a predetermined dead time. In other words, the electronic valve 69 will pass the first output pulse of one of the multipliers l8 and will thereafter remain insensitive for a predetermined length of time to any pulse from any one of the photomultipliers. The output of the electronic valve 69, in turn, is passed to an amplifier 74.

The output of the amplifier 74 is impressed on two pulse height discriminators 75 and 76 connected in parallel. Again, the two differential discriminators are arranged in such a way that they will only pass electrical pulses having a value between two predetermined limits. The output of the pulse height discriminator 75 is impressed on the amplifier 72 while that of the discriminator 76 is connected to the amplifier 73.

As a result, amplifier 72 will respond only if the size of the cell is within certain limits as determined by discriminator 70 while the size of its nucleus is within another pair of limits determined by the discriminator 75. Similarly, amplifier 73 responds to the two other sets of values of the cell volume and the size of the nucleus. [1 is to be understood that amplifiers 72 and 73 can readily be arranged to be responsive only to the size of the cell or only to the size of the nucleus.

These values, of course, will have to be set in accordance with experimental data some of which already exists as shown by the paper previously referred to.

It will readily be apparent that the cells can be sorted into one, two, or more different flasks depending on their characteristics by utilizing a smaller or larger number of discriminators such as 70, 71 and 75, 76.

Accordingly, the size of the nucleus and the size of the cytoplasm may be determined in one operation and entirely automatically. Such an operation, of course. requires much less time than if it were done by a person. The result of this segregation of the celk is that the number of suspicious or atypical cells can be vastly increased so that there is a greater chance and a greater certainty of diagnosing cancer if such exists. Also, this makes it possible to review or check the enriched atypical cells by a cytologist. It will, of course, be realized since the machine only classifies suspicious cells that in many instances the cells clearly indicates that there is no cancer. 0n the other hand, it is also possible to find cells exfoliated from the abnormal cervix which are usually called dysplasic. Normal cells, dysplasic cells, and cells from an in situ carcinoma have significant differences in the sizes of their nuclei and cytoplasms.

This has been described in a paper by George L. Wied, Gildardo Legorreta, Dietrich Mohr, and Andre Rauzy, which was published in Vol. 97 of the Annals of the New York Academy of Sciences, page 759 September 29, 1962). Such dysplasic cells are interpreted by some researchers as an indication of a pre-cancerous stage. Thus, any patient who who is found to have dwplasic cells can be carefully watched in subsequent years to improve the chances of finding cancer in its earliest stages.

it might be noted that it is relatively easy to dilute the cells in such a way to make sure that the selected cells are sucked into their respective conduits such as 38 or 40. Thus, assuming that there are four cells per cubic millimeter, this corresponds to four million cells in 1 liter. Such a concentration is sufficiently diluted to insure that each wanted cell is removed into its proper conduit. While, at the same time, insuring a minimum contamination by non-wanted cells. On the other hand, there are enough cells in the suspension to make sure that the sample being investigated is representative.

it should also be noted that the criteria are frequently different for different types of cancer. Hence, the pulse height discriminators, such as 70, 71, and 75,76, can be arranged in such a manner that they will collect all cells which are not normal, that is, all atypical cells and that the atypical cells, in turn, are segregated in accordance with still other characteristics.

Preferably, the size of the aperture is adjusted by the knife edges 51 and 52 in accordance with the size of the cells or groups of cells which are being classified. The range of the size of the cells or group of cells is from 5 to 200 microns. Preferably, the aperture is about ten times the diameter of the cells or groups of cells. Furthermore, the direction of the aperture is preferably perpendicular to the main diameter of the unmasked portion of the diaphragms 19.

It will also be evident how the apparatus of FIGS. 1 to 3 can be used for counting at the same time both red and white blood corpuscles in a suitable suspension. in the first place, the total number of corpuscles is counted by the amplifier 65. Those which have a nucleus, namely, the white blood corpuscles, are counted by the amplifier 74. Hence, subtracting one from the other, it is readily apparent that both the number of red and of white blood corpuscles can be counted simultaneously. However, there is one exception, namely, in some rare cases, nucleated red blood cells may be found in the blood stream. if desired, a conventional electronic counter such as shown at 77 may be coupled to the output of each of the amplifiers 65 and 74 thereby to count the number of cells and the number of nuclei which pass through the focal plane of the microscope. It is also feasible to connect a counter such as shown at 78 to the output of each amplifier 72 and 73 to count the number of cells with certain characteristics to be collected in flasks 34 and 35.

It may be noted that a magnetic field is established within the liquid rather than an electric current. In accordance with the prior art, an electric current is set up which, in turn, produces heat. Hence, the apparatus can be used for less than a minute at a time. The magnetic or static field produced in accordance with the present invention does not primarily produce an electric current and, hence, the heat is minimized. The magnetic field is disturbed by the movement of the liquid unless the liquid is non-conductive and does not have any magnetic dipoles. However, the disturbance of the magnetic field in proportion to the velocity of the liquid provides a means of measuring the volume of liquid that has passed. Furthermore, of course, the magnetic field is disturbed by the occurrence of a particle of cell as explained above.

instead of producing a magnetic field, it is also feasible to produce a steady electric field as illustrated in FIG. 5. Thus, there is illustrated a tube 80 which represents any conduit through which suspended particles are flowing in the direction of arrows 81. A pair of insulated metallic electrodes 82 and 83 are disposed at the end of another, preferably, insulating cylinder or conduit 84. These electrodes 82 and 83 may be connected to a source of electric potential by leads 85 and 86, to provide an electric capacitor.

Accordingly, every time a cell passes through the metal electrodes 82, 83, the electric field is varied. This variation can be picked up either directly by the electrodes 82, 83 or by another suitable pair of electrodes. The cells may then be sucked up by the conduit 84 which may be connected to a vacuum by means of valves in the manner explained in connection with FIG. 1, or may be allowed to continue to flow through the conduit 8|.

The devices of FIGS. or 6 may also be arranged as a small pipette which is moved by hand through a container with a cell suspension for picking up particular cells.

As explained hereinbefore it is also feasible to utilize ultraviolet light which is absorbed by a nucleus so that the cell nucleus does not have to be stained. To this end, the arrangement of H6. 4 may be used with advantage. Instead of using an optical lens system, which is transparent to ultraviolet light, such as quartz or fluorite, it may be more practical to utilize a reflecting system rather than a refracting system.

Thus, the compound microscope of H6. 4 includes various light reflecting lenses or surfaces 90 and 91 which may form the objective I6, 92 and 93 which may form the ocular l7 sealed by a quartz plate 89. The light source and condenser lens have been omitted. A cell 93 (not drawn to scale) is shown at the focal plane of the microscope. The light from the microscope is projected into the photomultiplier tube 18 which should have a photocathode responsive to ultraviolet light.

The suspended cells are housed in the container 20 which may be connected directly to the chamber 22 of the microscope. Since there are no refracting lenses the entire space between the objective l6 and the ocular l7 and up to the quartz plate 89 is open for the liquid.

It will be understood, of course, that the cells are again classified in accordance with the size or volume of the cytoplasm and the size of the nucleus in the manner previously explained by means of the arrangement illustrated in FIG. 1.

It should be noted that it is also feasible to use a microscope as shown in FIGS. 1 or 4 permitting a dark field illumination.

The arrangement of FIG. 6 to which reference is now made, again shows a glass tube or pipette 80 having a number of outlets 31, 38, and 40 which may be controlled in the manner previously explained. The glass tube 80 is surrounded by the suspension containing the particles to be classified. Thus, the glass tube 80 may be disposed in a container 96 having a suitable bottom wall 99. The liquid enters the tube 80 at the narrow top. The tube 80 is provided with two electrodes 97 and 98 which are connected, for example, to the two poles of a bat tery. These two electrodes 97 and 98 establish an electric field through the opening of tube 80. Hence, when a particle or cell crosses the opening of the tube the electric field between the two electrodes 97 and 98 is varied. This, in turn, can be detected and amplified either by electrodes 97, 98 or by additional pick-up electrodes in the manner previously explained.

FIG. 7 illustrates a light condenser which may be used in lieu of a condenser lens illustrated in FIGS. 1 and 3. The lens arrangement of FIG. 7 permits to illuminate any object in the focal plane both by direct and tangential light. To this end, the condenser system includes a central lens system 100 and a peripheral or annular lens system 103 separated by an opaque cylinder 101. Each lens system 101 and 103 has its own light source (not shown). The central lens system I00 is substantially an objective lens in an inverted position. Such an arrangement is known for creating an illumination in a small area.

The peripheral lens system 103 consists substantially of an ordinary light condenser provided with a central aperture in which the central lens system is disposed. The peripheral lens system 103 provides lateral illumination of the focal plane or field thus to provide what is generally called a dark field illamination.

It should be noted that generally the light condenser used for dark field illumination has a dark diaphragm disposed between the light source and the lens. This serves the purpose to eliminate light in the central area so that only the peripheral lens area is used. The same result is obtained with the arrangement of FIG. 7 except that the space not normally used is now utilized by the provision of the central lens system 100.

However, it will be understood that the peripheral lens system 103 may be used by itself to provide lateral illumination or dark field illumination. This may be done, for example, where the microscope is to be connected to a continuous flow so that the central aperture can be used to provide an outlet for the gas or liquid.

The arrangement of FIG. 7 is preferably used in connection with a feedback system so that the lens systems 100 and 103 can be operated alternatively. Accordingly, when a particle crosses the dark field and, hence, scatters the light, the output of the photomultiplier may be used for turning off momentarily the light source associated with the peripheral lens system 103. At the same time, the light source is turned on for the central lens system 100. Thereupon, the output of the photomultiplier can be used again to turn 05 the light for the central lens system 100 and turn on the light of the outer lens system 103.

It will be appreciated that the structure of H6. 7 permits to determine two different characteristics of a particle to be classified such as the total size of a cell and the size of the nucleus by means of both scattered and transmitted light. Hence, it is not necessary to utilize either a steady magnetic or steady electric field although the provision of such a field may be preferred.

it should be noted that the above described arrangement may be utilized for classifying particles suspended in a gas such, for example, as air. Also, the above'described apparatus may be connected in a pipe through which there is a continuous flow of a liquid or gas having suspended particles therein. These particles may then be counted, classified, or segregated for further study in accordance with the teachings of this invention.

There has thus been disclosed a method of and apparatus for classifying microscopic particles such as cells suspended in a liquid and for sorting them in accordance with predetermined characteristics. For example, it is possible to measure both the size of the cell and the size of its nucleus. It is further possible to classify and to segregate cells in accordance with certain predetermined values of the cell size and nucleus size. This operation takes place automatically.

Hence, it is possible to vastly increase the number of suspicious or atypical cells in a sample of cells. This, in turn, should greatly facilitate diagnosis of cancer and the like. It is also possible to utilize this apparatus for determining the effects of hormones on the vaginal epithelium or the function of the ovary. Another application of the method of and apparatus of the present invention is for counting simultaneously both red and white blood cells without the necessity of using separate dilutions for the red cells and for the white cells. Also, it is feasible to classify the white blood cells according to the size of the nucleus and the color and size of the cytoplasm, provided that the leucocytes have previously been differentially stained.

l claim:

Apparatus for investigating microscopic particles suspended in a transparent fluid, comprising:

a microscope having a focal plane;

means for causing the suspension of particles to flow in a path, said path extending from one side and across said focal plane to the other side thereof;

means for illuminating said focal plane and means for measuring the variation of the light caused by absorption of the light by each particle as it flows through said focal plane;

means for establishing a steady electromagnetic field extending through said focal plane; and

means for measuring the change of the electromagnetic field caused by the passage of a particle through said focal plane.

Apparatus for classifying microscopic particles suspended in a transparent liquid, comprising:

a microscope having a focal plane; means for causing the suspension of particles to flow in a path, said path extending from one side to the other side and once across said focal plane; means for illuminating said focal plane; means for detecting changes of the light absorbed by each particle as it flows through said focal plane; and means disposed adjacent said focal plane for selectively removing a desired particle and collecting it.

Apparatus for classifying microscopic cells having a nucleus and suspended in a transparent liquid, comprising:

a microscope having a focal plane;

means for causing the suspension of cells to flow in a path, said path crossing said focal plane once;

means for illuminating said focal plane and for measuring the light absorbed by each cell as it flows through said focal plane, thereby to determine the image of the cell;

. means for establishing a steady electromagnetic field extending through said focal plane;

. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane, thereby to trigger the light absorption measuring means; and

means for detecting the cells that have predetermined image characteristics.

Apparatus for investigating microscopic cells having a nucleus and suspended in a transparent liquid, comprising:

a microscope having an objective providing a focal plane, said objective being immersed in the liquid;

means for causing the suspension of cells to flow in a path, said path extending from one side across said focal plane and to the other side thereof;

. means for illuminating said focal plane with light of a wave length absorbed by the nucleus of the cells;

. means for detecting and measuring the light absorbed by the nucleus of each cell as it flows through said focal plane;

means for establishing a steady electromagnetic field extending through said focal plane; and

means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane to determine the volume of the cell.

5. Apparatus for classifying microscopic cells having a nucleus and suspended in a transparent liquid, comprising:

a microscope having an objective providing a focal plane, said objective being immersed in the liquid;

means for causing the suspension of cells to flow in a path, said path extending once across said focal plane; means for illuminating said focal plane with light of a wave length absorbed by the nucleus of the cells;

means for detecting and measuring the light absorbed by the nucleus of each cell as it flows through said focal plane;

. means for establishing a steady electromagnetic field ex tending through said focal plane;

means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane to determine the volume of the cell; and

means disposed adjacent said focal plane for selectively removing a desired cell and for collecting it.

Apparatus for counting simultaneously both red and white blood corpuscles suspended in a transparent liquid, the white blood corpuscles having each a nucleus, said apparatus comprising:

. means for establishing a steady electromagnetic field extending through said focal plane;

. means for measuring the change of the electromagnetic field caused by the passage of a blood corpuscle through said focal plane, thus enabling the detection of the total number of blood corpuscles; and

means for counting the total blood corpuscles and including the white blood corpuscles.

Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a compound microscope having an objective immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

means for causing the suspension of cells to flow across said focal plane;

means for illuminating said focal plane with light having a wave length range absorbed by the nucleus of the cells of said suspension;

means including a light sensitive means disposed adjacent the ocular of said microscope and responsive to the variation of light caused by the nucleus of each cell as it moves across said focal plane;

means for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

hydraulic pressure means disposed adjacent said focal plane for selectively removing a desired cell and collectin g it; and

h. means responsive to a predetermined range of values of the light absorbed by the nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined size from other cells.

Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a compound microscope having an objective immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane; means for illuminating said focal plane with light having a wave length range absorbed by the nucleus of the cells of said suspension;

. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of light caused by the absorption of light by each cell as it moves across said focal plane;

means for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

. means disposed adjacent said focal plane for selectively a transparent liquid to provide a suspension of cells, said apparatus comprising:

a compound microscope having an objective, said objective being immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

. means for causing the suspension of cells to flow across said focal plane;

. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

. an opaque diaphragm having a variable slit disposed in said focal plane;

means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by the nucleus of each cell as it moves across said focal plane;

. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

. means responsive to a predetermined range of values of the variation of light caused by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

10. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a compound microscope having an objective said objec tive being immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

. means for causing the suspension of cells to flow across said focal plane;

. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

means including a light sensitive means disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by at least a portion of each cell as it moves across said focal plane;

. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

. means responsive to a predetermined range of values of the variation of light caused by a cell for actuating said means for removing a cell, thereby to segregate cells havin g predetermined light absorption from other cells.

11. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a compound microscope having an objective, said objective being immersed in the liquid, and an ocular and providing a focal plane adjacent said objective;

said microscope having a central annular light condenser and an outer annular light condenser spaced therefrom, both being immersed in the liquid for illuminating said focal plane by direct and tangential light;

means for causing the suspension of cells to flow across said focal plane;

means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by the nucleus of each cell as it moves across said focal plane;

means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

means responsive to a predetermined range of values of the variation of light caused by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

12. An attachment for a compound microscope of the type having an objective and ocular and providing a focal plane adjacent said objective, said attachment comprising:

means for causing a suspension of cells to flow across said focal plane;

b. means for illuminating the microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

means including a light sensitive device disposed adjacent the ocular of the microscope and responsive to the variation of light absorbed by the nucleus of each cell as it moves across said focal plane;

means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

means responsive to a predetermined range of values of the light absorbed by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

13. Apparatus for classifying microscopic particles suspended in a fluid to provide a suspension of particles, said apparatus comprising:

means for establishing a steady electromagnetic field within a predetermined volume of a liquid;

means for measuring the change of the field caused by the passage of each individual particle through said volume and indicative of the volume of the particle;

means disposed in the vicinity of said volume for selectively removing a desired particle and collecting it; and means responsive to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a particle, thereby to segregate panicles having a predetermined volume from other particles.

14. Apparatus for classifying microscopic particles suspended in a fluid to provide a suspension of particles, said apparatus comprising:

means for establishing a steady electromagnetic field within a predetermined volume of a liquid;

. means for measuring the change of the field caused by the passage of each individual particle through said volume and indicative of the volume of the particle;

. means disposed in the vicinity of said volume for selectively removing a desired particle and collecting it;

means responsive to a predetennined range of values of the change of the electromagnetic field for actuating said means for removing a particle, thereby to segregate panicles having a predetermined volume from other particles; and

means for counting the collected particles.

15. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a compound microscope having a reflective objective and a reflective ocular immersed in the liquid and providing a focal plane adjacent said objective;

means including a pressure differential for causing the suspension of cells to flow across said focal plane;

means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

. means including a photomultiplier disposed adjacent the ocular of said microscope and responsive to the amount of light absorbed by the nucleus of each cell as it moves across said focal plane;

means for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell acros said focal plane and indicative of the volume of the cell;

g. means disposed adjacent said focal plane for developing a pressure differential to selectively remove a desired cell and collect it; and

h. means responsive to a predetermined range of values of the light absorbed by a nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined volume from other cells.

16. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. means including a photocathode disposed adjacent the ocular of said microscope and responsive to the amount of light absorbed by the nucleus of each cell as it moves across said focal plane;

. means including a capacitor for establishing a steady electromagnetic field within a volume including said focal plane; means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

. means responsive to a predetermined range of values of the light absorbed by a nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined volume from other cells.

17. A method of investigating microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. providing a restricted testing passage;

c. adjusting the size of said testing passage depending upon the size of the particles to be measured and to assure an unclogged condition;

d. causing the suspended particles to flow past said adjusted testing passage;

e. providing a steady electromagnetic field within said testing passage; and

f. measuring the change of said electromagnetic field caused by the passage of each individual particle through said testing passage, thereby to determine the volume of the particle.

18. A method of investigating microscopic particles which comprises the steps of:

comprises the steps of:

a. suspending the particles to be classified in a liquid; b. causing the suspended particles to flow past a predetermined testing volume;

c. establishing an electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of each individual particle through the testing volume, thereby to determine one characteristic of the particle;

e. simultaneously projecting light through the particle; and f. detecting at least another characteristic of the particle from the absorption of light passing through the particle. 20. A method of classifying cells which comprises the steps a. suspending the cells to be classified in a liquid;

b. causing the suspended cells to flow past a predetermined testing volume;

c. establishing an electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of a cell through the testing volume thereby to determine the volume of the cell;

e. simultaneously illuminating the testing volume; and

f. measuring the light absorbed by the cell within the testing volume, thereby to detect at least one characteristic of the cell from the light absorbed by the cell.

21. A method of classifying microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. causing the suspended particles to flow past a predetermined testing volume;

c. projecting light through the particle in the same direction of the particle flow;

d. detecting at least one characteristic of the particle from the light absorbed by passing through the particle; and

e. separating the particle in accordance with said characteristic.

22. A method of classifying microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. causing the suspended particles to flow past a predetermined testing volume;

c. establishing an electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of a particle through the testing volume, thereby to determine one characteristic of the particle;

e. projecting light through the particle;

f. detecting at least another characteristic of the particle from the absorption of the light passing through the particle; and

g. separating the particles in accordance with both of said characteristics.

23. A method of automatically counting simultaneously red and white blood cells suspended in a transparent liquid comprising the steps of:

a. staining the white blood cells;

b. causing the blood cells to fiow through a predetermined focal plane;

c. projecting light through said focal plane having a wave length which is absorbed by the stained white blood cells;

d. measuring the light absorbed by each white cell passing through said focal plane;

e. establishing an electromagnetic field in a volume including said focal plane; and

f. measuring the change of the electromagnetic field caused by the movement of a blood cell through said focal plane thereby to count the total number of blood cells and the number of white blood cells.

24. A method of automatically classifying white blood cells suspended in a transparent liquid comprising the steps of:

a. staining the nuclei and the cytoplasm of the white blood cells;

b. causing the white blood cells to flow through a predetermined focal plane;

c. projecting light through said focal plane;

comprising the steps of:

a. digesting the intercellular substance;

b. suspending the cells in a transparent liquid;

c. staining the nucleus and the cytoplasm of the cells;

d. causing the cells to flow once across a focal plane;

e. projecting light on said focal plane;

f. detecting the change in the intensity of light caused by a cell;

g. detecting the color of the image;

h. establishing an electromagnetic field in a volume including said focal plane;

i. detecting the change of the electromagnetic field caused by the particle when crossing the focal plane, thereby to determine one characteristic of the particle; and

j. separating and counting the particles in accordance with said characteristics.

26. A method of automatically classifying biological cells comprising the steps of:

a. digesting the intercellular substance;

b. suspending the cells in a liquid;

. eliminating the clusters of cells;

. differentially staining the nucleus;

. suspending the cells in a transparent liquid;

causing the cells to flow across a focal plane so that each cell is momentarily exactly in said focal plane;

projecting light on said focal plane;

. detecting and measuring the change in the light caused by the nucleus;

. establishing an electromagnetic field in a volume including said focal plane;

j. detecting and measuring the change in the electromagnetic field caused by the passage of a cell through said focal plane; and

. separating the cells in accordance with the light change and the change in the electromagnetic field.

27. A method of separating microscopical particles in a microscope having a focal plane, comprising the steps of:

a. suspending the particles in a transparent liquid;

b. causing a flow of liquid through the focal plane;

c. illuminating the focal plane;

d. measuring the variation in the light produced by the particle; and

e. producing a reduction of pressure in the suspending liquid to aspirate the said particle.

28. A method of separating and classifying particles by their size comprising the steps of:

a. suspending the particles in a liquid;

b. causing a flow of the-liquid;

c. producing an electromagnetic field in the liquid;

d. sizing the particles by the change in the electromagnetic field, and

e. producing a reduction of ambient liquid pressure to cause a selected particle to flow in a desired direction for collecting it.

29. Apparatus for classifying leucocytes which have previnear:

ously been stained and which are suspended in a transparent liquid, said apparatus comprising:

a. a compound microscope having an objective, said objective being immersed in the liquid and a plurality of oculars, each receiving light from said objective, said microscope providing a focal plane adjacent said objective;

b. means for causing the suspended leucocytes to flow across said focal plane;

c. means for illuminating said focal plane with light having a wave length range absorbed by stained leucocytes; d. means including a color filter and light sensitive means disposed adjacent each ocular of said microscope and 5 responsive to the variation of light caused by light absorption by at least a portion of a leucocyte as it moves across said focal plane; e. means disposed adjacent said focal plane for selectively removing a desired leucocyte and collecting it; means responsive to a predetermined range of values of the variation of light absorption caused by a leucocyte for actuating said means for removing a leucocyte, thereby to segregate leucocytes having predetermined color absorption from other leucocytes; and

g. means for counting the leucocytes in each predetermined range of values.

30. A method of investigating particles by their size, comprising the steps of:

a. suspending the particles in a liquid;

b. moving the particles through a testing passage;

c. adjusting the size of said testing passage in accordance with the size of the particles to be measured and to assure an unclogged condition;

d. sizing the particles by subjecting to an applied field; and

e. triggering one or more actions when the size of a particle is in a range of predetermined values.

31. An apparatus for classifying particles suspended in a fluid, to provide a suspension of particles, said apparatus comprising:

a. a testing passage of a restricted size;

b. means for adjustably increasing and decreasing the size of said testing passage in proportion to the size of the particle to be measured, and relieve any existing clogging;

c. means for causing the suspension of particles to cross through said testing passage;

d. means for providing an applied field within said testing passage; and

e. means for detecting the change of the field caused by the passage of each particle through said testing passage.

32. A method of investigating particles suspended in a liquid which comprises the steps of:

a. causing the suspended particles to cross past a predetermined testing volume;

b. establishing a steady electric field within the testing volume;

c. detecting the change of the field caused by the passage of a particle through the testing volume; and

d. separating the desired particles from the particle suspension, according to predetermined values of said change of the field.

33. An apparatus for classifying particles suspended in a fluid to provide a suspension of particles, said apparatus comprising:

a. a testing passage of a restricted size;

b. means for adjustably increasing and decreasing the size of said testing passage in proportion to the size of the particle to be detected;

c. means for causing the suspension of particles to cross through said testing passage;

d. means for providing a magnetic field within said testing passage;

e. means for detecting the change of the field caused by the passage of each particle through said testing passage; and

t. means for classifying the particles in response to a predetermined range of values of the change of the magnetic field.

34. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a fluid;

b. causing the suspended particles to cross through a testing passage;

c. adjusting the size of said testing passage in accordance with the size of the particles to be investigated and to as sure an unclogged condition;

d. providing a magnetic field within said testing passage; and

e. measuring the size of the particles by detecting the change in the field caused by the passage of a particle through said testing passage.

35. An apparatus for investigating particles suspended in a fluid, to provide a suspension of particles, said apparatus comprising:

a. a testing volume restricted as to size according to the size of the particles to be investigated;

b. means for adjusting the size of said testing volume;

c. means for causing the suspension of particles to cross through said testing volume;

d. means for establishing a steady electric field within the testing volume;

e. means for measuring the change of the capacitance of said electric field caused by the passage of each particle through said testing volume;

f. means for establishing a steady magnetic field within the testing volume; and

g. means for measuring the change of the permeability of said magnetic field caused by the passage of each particle through said testing volume.

36. A combination as defined in claim 35 including means for classifying the particles according to predetermined values of changes of the electric and the magnetic fields.

37. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a fluid;

b. causing the suspended particles to cross past a testing volume;

c. changing the size of the testing volume according to the size of the particles to be investigated and to provide an unclogged condition;

d. establishing a steady electric field within the testing volume;

e. measuring the change of the capacitance of said electric field caused by the passage of each particle through said testing volume;

. establishing a steady magnetic field within the testing volume; and

. measuring the change of the permeability of said magnetic field caused by the passage of each particle through said testing volume.

38. An apparatus for studying the dielectric properties of fluids comprising:

a. a testing passage;

b. means for adjusting in position the size of said testing passage;

c. means to cause a sample of the fluid to flow through said testing passage;

d. means to produce a steady electric field within said testing passage;

e. means to detect the change in capacitance of said field by said fluid; and

f. means for diverting a portion of the fluid according to predetermined values of the capacitance change.

39. Apparatus for investigating particles suspended in a liquid comprising:

a. a testing volume;

b. means for causing the suspension of particles to cross through said testing volume;

c. means for establishing a magnetic field within the testing volume to induce an electrical field in the flowing liquid;

d. means, including a pair of pick-up electrodes, for detecting the change of the induced electrical field caused by the passage of each particle through said testing volumne; and

e. means for classifying the particles according to predetermined values of the change detected by said pick-up electrodes.

40. An apparatus for investigating particles, such as cells,

suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means, including an opaque diaphragm with an aperture, which coincide with said focal plane, for causing the suspension of cells to flow through said focal plane;

c. means for changing the size of the focal plane;

d. means for illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. means for detecting the image produced by each particle;

f. means, including a light-proof shield, for preventing stray light reaching the image detecting means;

g. means for producing an electrical current through said focal plane; and

h. means for measuring the variations produced in the electrical current by each particle, as it flows through said focal plane, and thereby determining the size of the particle.

41. A combination as defined in claim 40 including means for classifying the particles according to predetermined values of the measured characteristics.

42. A combination as defined in claim 41 including means responsive to a predetermined range of variations of the light caused by at least a portion of the cell, and to a predetermined range of values of the change of the electrical current for actuating said means for classifying said particles.

43. A combination as defined in claim 41 including means disposed in the vicinity of said testing volume for removing the desired particles from the particle suspension.

44. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a transparent liquid;

b. causing the suspended particles to flow through the focal plane of a microscope, said focal plane having an opaque diaphragm with an aperture;

c. changing the size of said aperture in accordance with the size of the particles to be investigated and to assure an unclogged condition;

d. illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. detecting the image produced by each particle;

f. shielding the image detection against stray-light;

g. producing an electrical current through said focal plane;

and

h. measuring the variations in the electrical current produced by each particle as it flows through said focal plane, and thereby determining the size of the particle.

45. A method of studying biological cells suspended in a liquid, comprising the steps of:

a. measuring the size of the nucleus by the ultraviolet light absorbed by the cell, and at the same time producing an image of the cell;

b. measuring the size of the cytoplasm by the disturbance produced in different applied fields by the same cell;

c. determining the ratio of nucleus size to cytoplasm size in each cell; and

d. multiplying the number of cell images when the nucleuscytoplasm ratio is within a predetermined range of values, and measuring the amount of light absorbed by a number of areas in which the cell is divided.

46. An apparatus for investigating particles suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means for causing the suspension of particles to flow through said focal plane;

c. means for illuminating said focal plane with light absorbed by the particles to thereby produce an image;

d. means for measuring the image density, therefore determining the absorption of light produced by a particle;

e. means for sizing the particles as they cross said focal plane, thereby triggering the image density-measuring means when the size of a particle is between predetermined values, and

f. means responsive to predetermined image characteristics and to a predetermined range of cell-size for removing a cell from the suspension.

47. An apparatus for investigating particles, such as cells. 75 suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means, including an opaque diaphragm with an aperture, which coincide with said focal plane, for causing the suspension of cells to flow through said focal plane;

c. means for changing the size of the focal plane;

d. means for illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. means for detecting the image produced by each particle;

f. means, including a light-proof shield, for preventing stray light reaching the image detecting means;

g. means for producing a magnetic field through said focal plane to induce an electrical field in the liquid flowing through said focal plane; and

b. means, including a pair of pick-up electrodes. for measuring the variations produced in the electrical field by each particle, as it flows through said focal plane, and thereby determining the size of the particle.

48. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a transparent liquid;

b. causing the suspended particles to flow through the focal plane of a microscope, said focal plane having an opaque diaphragm with an aperture;

c. changing the size of said aperture in accordance with the size of the particles to be investigated and to assure an unclogged condition;

. illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

c. detecting the image produced by each particle;

f. shielding the image detection against stray light;

g. producing an electrical field through said focal plane; and

h. measuring the variations in the electrical field produced by each particle as it flows through said focal plane, and thereby determining the size of the particle.

49. An improvement for an apparatus for investigating particles suspended in a liquid, said apparatus having means for producing an electrical current through a testing volume, said improvement comprising:

a. means for measuring the variation produced in the electrical current, by each particle, as it flows through the testing volume, and thereby determining its size;

b. a microscope having a focal plane which coincides with said testing volume;

c. means for changing the size of the testing volume;

d. means for illuminating said focal plane with light that is absorbed by a particle as it flows through said focal plane, and thereby produce an image of the particle; and

e. means for detecting the image produced by each particle.

50. A method of studying biological cells suspended in a liquid comprising the steps of:

a. producing an image of a cell through a microscope;

b. dividing the image of the cell in a number of areas;

c. measuring the light in each one of said areas;

d. measuring the size of the nucleus by the number of areas in which the amount of light reaches a predetermined threshold;

e. measuring the size of the cytoplasm by the disturbance produced in different applied mediums by the same cell;

f. determining the ratio of nucleus size to cytoplasm size;

and

g. triggering one or more actions when the ratio of nucleuscytoplasm is within predetermined range of values.

t t l i i 

2. Apparatus for classifying microscopic particles suspended in a transparent liquid, comprising: a. a microscope having a focal plane; b. means for causing the suspension of particles to flow in a path, said path extending from one side to the other side and once across said focal plane; c. means for illuminating said focal plane; d. means for detecting changes of the light absorbed by each particle as it flows through said focal plane; and e. means disposed adjacent said focal plane for selectively removing a desired particle and collecting it.
 3. Apparatus for classifying microscopic cells having a nucleus and suspended in a transparent liquid, comprising: a. a microscope having a focal plane; b. means for causing the suspension of cells to flow in a path, said path crossing said focal plane once; c. means for illuminating said focal plane and for measuring the light absorbed by each cell as it flows through said focal plane, thereby to determine the image of the cell; d. means for establishing a steady electromagnetic field extending through said focal plane; e. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane, thereby to trigger the light absorption measuring means; and f. means for detecting the cells that have predetermined image characteristics.
 4. Apparatus for investigating microscopic cells having a nucleus and suspended in a transparent liquid, comprising: a. a microscope having an objective providing a focal plane, said objective being immersed in the liquid; b. means for causing the suspension of cells to flow in a path, said path extending from one side across said focal plane and to the other side thereof; c. means for illuminating said focal plane with light of a wave length aBsorbed by the nucleus of the cells; d. means for detecting and measuring the light absorbed by the nucleus of each cell as it flows through said focal plane; e. means for establishing a steady electromagnetic field extending through said focal plane; and f. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane to determine the volume of the cell.
 5. Apparatus for classifying microscopic cells having a nucleus and suspended in a transparent liquid, comprising: a. a microscope having an objective providing a focal plane, said objective being immersed in the liquid; b. means for causing the suspension of cells to flow in a path, said path extending once across said focal plane; c. means for illuminating said focal plane with light of a wave length absorbed by the nucleus of the cells; d. means for detecting and measuring the light absorbed by the nucleus of each cell as it flows through said focal plane; e. means for establishing a steady electromagnetic field extending through said focal plane; f. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane to determine the volume of the cell; and g. means disposed adjacent said focal plane for selectively removing a desired cell and for collecting it.
 6. Apparatus for counting simultaneously both red and white blood corpuscles suspended in a transparent liquid, the white blood corpuscles having each a nucleus, said apparatus comprising: a. a microscope having a focal plane; b. means for causing the blood corpuscles to flow in a path, said path passing once through said focal plane; c. means for illuminating said focal plane both with direct and indirect light; d. means for measuring the light absorbed by the nucleus of each white blood corpuscle as it flows through said focal plane, thus enabling the detection of each white blood corpuscle; e. means for establishing a steady electromagnetic field extending through said focal plane; f. means for measuring the change of the electromagnetic field caused by the passage of a blood corpuscle through said focal plane, thus enabling the detection of the total number of blood corpuscles; and g. means for counting the total blood corpuscles and including the white blood corpuscles.
 7. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having an objective immersed in the liquid and an ocular and providing a focal plane adjacent said objective; b. means for causing the suspension of cells to flow across said focal plane; c. means for illuminating said focal plane with light having a wave length range absorbed by the nucleus of the cells of said suspension; d. means including a light sensitive means disposed adjacent the ocular of said microscope and responsive to the variation of light caused by the nucleus of each cell as it moves across said focal plane; e. means for establishing a steady electromagnetic field within a volume including said focal plane; f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell; g. hydraulic pressure means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and h. means responsive to a predetermined range of values of the light absorbed by the nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined size from other cells.
 8. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having an objective immersed in the liquid and an ocular and providing a focal plane adjacent said objective; b. means for causing the suspension of cells to flow across said focal plane; c. means for illuminating said focal plane with light having a wave length range absorbed by the nucleus of the cells of said suspension; d. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of light caused by the absorption of light by each cell as it moves across said focal plane; e. means for establishing a steady electromagnetic field within a volume including said focal plane; f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell; g. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and h. means responsive to a predetermined range of values of the variation of the light caused by at least a portion of the cell and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined size from other cells.
 9. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having an objective, said objective being immersed in the liquid and an ocular and providing a focal plane adjacent said objective; b. means for causing the suspension of cells to flow across said focal plane; c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension; d. an opaque diaphragm having a variable slit disposed in said focal plane; e. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by the nucleus of each cell as it moves across said focal plane; f. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and g. means responsive to a predetermined range of values of the variation of light caused by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.
 10. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having an objective, said objective being immersed in the liquid and an ocular and providing a focal plane adjacent said objective; b. means for causing the suspension of cells to flow across said focal plane; c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension; d. means including a light sensitive means disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by at least a portion of each cell as it moves across said focal plane; e. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and f. means responsive to a predetermined range of values of the variation of light caused by a cell for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.
 11. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having an objective, said objective being immersed in the liquid, and an ocular and providing a focal plane adjacent said objective; b. said microscope having a central annular light condenser and an outer annular light condenser spaced therefrom, both being immersed in the liquid for illuminating said focal plane by direct and tangential light; c. means for causing the suspension of cells to flow across said focal plane; d. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension; e. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by the nucleus of each cell as it moves across said focal plane; f. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and g. means responsive to a predetermined range of values of the variation of light caused by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.
 12. An attachment for a compound microscope of the type having an objective and ocular and providing a focal plane adjacent said objective, said attachment comprising: a. means for causing a suspension of cells to flow across said focal plane; b. means for illuminating the microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension; c. means including a light sensitive device disposed adjacent the ocular of the microscope and responsive to the variation of light absorbed by the nucleus of each cell as it moves across said focal plane; d. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and e. means responsive to a predetermined range of values of the light absorbed by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.
 13. Apparatus for classifying microscopic particles suspended in a fluid to provide a suspension of particles, said apparatus comprising: a. means for establishing a steady electromagnetic field within a predetermined volume of a liquid; b. means for measuring the change of the field caused by the passage of each individual particle through said volume and indicative of the volume of the particle; c. means disposed in the vicinity of said volume for selectively removing a desired particle and collecting it; and d. means responsive to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a particle, thereby to segregate particles having a predetermined volume from other particles.
 14. Apparatus for classifying microscopic particles suspended in a fluid to provide a suspension of particles, said apparatus comprising: a. means for establishing a steady electromagnetic field within a predetermined volume of a liquid; b. means for measuring the change of the field caused by the passage of each individual particle through said volume and indicative of the volume of the particle; c. means disposed in the vicinity of said volume for selectively removing a desired particle and collecting it; d. means responsive to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a particle, thereby to segregate particles having a predetermined volume from other particles; and e. means for counting the collected particles.
 15. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having a reflective objective and a reflective ocular immersed in the liquid and providing a focal plane adjacent said objective; b. means including a pressure differential for causing the suspension of cells to flow across said focal plane; c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension; d. means including a photomultiplier disposed adjacent the ocular of said Microscope and responsive to the amount of light absorbed by the nucleus of each cell as it moves across said focal plane; e. means for establishing a steady electromagnetic field within a volume including said focal plane; f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell; g. means disposed adjacent said focal plane for developing a pressure differential to selectively remove a desired cell and collect it; and h. means responsive to a predetermined range of values of the light absorbed by a nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined volume from other cells.
 16. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising: a. a compound microscope having an objective and an ocular and providing a focal plane adjacent said objective; b. means for causing the suspension of cells to flow across said focal plane; c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension; d. means including a photocathode disposed adjacent the ocular of said microscope and responsive to the amount of light absorbed by the nucleus of each cell as it moves across said focal plane; e. means including a capacitor for establishing a steady electromagnetic field within a volume including said focal plane; f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell; g. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and h. means responsive to a predetermined range of values of the light absorbed by a nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined volume from other cells.
 17. A method of investigating microscopic particles which comprises the steps of: a. suspending the particles to be classified in a liquid; b. providing a restricted testing passage; c. adjusting the size of said testing passage depending upon the size of the particles to be measured and to assure an unclogged condition; d. causing the suspended particles to flow past said adjusted testing passage; e. providing a steady electromagnetic field within said testing passage; and f. measuring the change of said electromagnetic field caused by the passage of each individual particle through said testing passage, thereby to determine the volume of the particle.
 18. A method of investigating microscopic particles which comprises the steps of: a. suspending the particles to be classified in a liquid having a dielectric constant different from that of the particles; b. causing the suspended particles to flow past a predetermined testing volume; c. establishing a steady electromagnetic field within the testing volume; d. measuring the change of the electromagnetic field caused by the passage of each individual particle through the testing volume, thereby to determine the volume of the particle; and e. separating the particles in accordance with their volumes.
 19. A method of investigating microscopic particles which comprises the steps of: a. suspending the particles to be classified in a liquid; b. causing the suspended particles to flow past a predetermined testing volume; c. establishing an electromagnetic field within the testing volume; d. measuring the change of the electromagnetic field caused by the passage of each individual particle through the testing volume, therEby to determine one characteristic of the particle; e. simultaneously projecting light through the particle; and f. detecting at least another characteristic of the particle from the absorption of light passing through the particle.
 20. A method of classifying cells which comprises the steps of: a. suspending the cells to be classified in a liquid; b. causing the suspended cells to flow past a predetermined testing volume; c. establishing an electromagnetic field within the testing volume; d. measuring the change of the electromagnetic field caused by the passage of a cell through the testing volume, thereby to determine the volume of the cell; e. simultaneously illuminating the testing volume; and f. measuring the light absorbed by the cell within the testing volume, thereby to detect at least one characteristic of the cell from the light absorbed by the cell.
 21. A method of classifying microscopic particles which comprises the steps of: a. suspending the particles to be classified in a liquid; b. causing the suspended particles to flow past a predetermined testing volume; c. projecting light through the particle in the same direction of the particle flow; d. detecting at least one characteristic of the particle from the light absorbed by passing through the particle; and e. separating the particle in accordance with said characteristic.
 22. A method of classifying microscopic particles which comprises the steps of: a. suspending the particles to be classified in a liquid; b. causing the suspended particles to flow past a predetermined testing volume; c. establishing an electromagnetic field within the testing volume; d. measuring the change of the electromagnetic field caused by the passage of a particle through the testing volume, thereby to determine one characteristic of the particle; e. projecting light through the particle; f. detecting at least another characteristic of the particle from the absorption of the light passing through the particle; and g. separating the particles in accordance with both of said characteristics.
 23. A method of automatically counting simultaneously red and white blood cells suspended in a transparent liquid comprising the steps of: a. staining the white blood cells; b. causing the blood cells to flow through a predetermined focal plane; c. projecting light through said focal plane having a wave length which is absorbed by the stained white blood cells; d. measuring the light absorbed by each white cell passing through said focal plane; e. establishing an electromagnetic field in a volume including said focal plane; and f. measuring the change of the electromagnetic field caused by the movement of a blood cell through said focal plane thereby to count the total number of blood cells and the number of white blood cells.
 24. A method of automatically classifying white blood cells suspended in a transparent liquid comprising the steps of: a. staining the nuclei and the cytoplasm of the white blood cells; b. causing the white blood cells to flow through a predetermined focal plane; c. projecting light through said focal plane; d. measuring the light absorbed by the nucleus of each white cell passing through said focal plane; e. detecting the color of the cytoplasm; f. establishing an electromagnetic field in a volume including said focal plane; and g. measuring the change of the electromagnetic field caused by the movement of a blood cell through said focal plane, thereby to determine characteristics of the white blood cells.
 25. A method of automatically classifying biological cells comprising the steps of: a. digesting the intercellular substance; b. suspending the cells in a transparent liquid; c. staining the nucleus and the cytoplasm of the cells; d. causing the cells to flow once across a focal plane; e. projecting liGht on said focal plane; f. detecting the change in the intensity of light caused by a cell; g. detecting the color of the image; h. establishing an electromagnetic field in a volume including said focal plane; i. detecting the change of the electromagnetic field caused by the particle when crossing the focal plane, thereby to determine one characteristic of the particle; and j. separating and counting the particles in accordance with said characteristics.
 26. A method of automatically classifying biological cells comprising the steps of: a. digesting the intercellular substance; b. suspending the cells in a liquid; c. eliminating the clusters of cells; d. differentially staining the nucleus; e. suspending the cells in a transparent liquid; f. causing the cells to flow across a focal plane so that each cell is momentarily exactly in said focal plane; g. projecting light on said focal plane; h. detecting and measuring the change in the light caused by the nucleus; i. establishing an electromagnetic field in a volume including said focal plane; j. detecting and measuring the change in the electromagnetic field caused by the passage of a cell through said focal plane; and k. separating the cells in accordance with the light change and the change in the electromagnetic field.
 27. A method of separating microscopical particles in a microscope having a focal plane, comprising the steps of: a. suspending the particles in a transparent liquid; b. causing a flow of liquid through the focal plane; c. illuminating the focal plane; d. measuring the variation in the light produced by the particle; and e. producing a reduction of pressure in the suspending liquid to aspirate the said particle.
 28. A method of separating and classifying particles by their size comprising the steps of: a. suspending the particles in a liquid; b. causing a flow of the liquid; c. producing an electromagnetic field in the liquid; d. sizing the particles by the change in the electromagnetic field, and e. producing a reduction of ambient liquid pressure to cause a selected particle to flow in a desired direction for collecting it.
 29. Apparatus for classifying leucocytes which have previously been stained and which are suspended in a transparent liquid, said apparatus comprising: a. a compound microscope having an objective, said objective being immersed in the liquid and a plurality of oculars, each receiving light from said objective, said microscope providing a focal plane adjacent said objective; b. means for causing the suspended leucocytes to flow across said focal plane; c. means for illuminating said focal plane with light having a wave length range absorbed by stained leucocytes; d. means including a color filter and light sensitive means disposed adjacent each ocular of said microscope and responsive to the variation of light caused by light absorption by at least a portion of a leucocyte as it moves across said focal plane; e. means disposed adjacent said focal plane for selectively removing a desired leucocyte and collecting it; f. means responsive to a predetermined range of values of the variation of light absorption caused by a leucocyte for actuating said means for removing a leucocyte, thereby to segregate leucocytes having predetermined color absorption from other leucocytes; and g. means for counting the leucocytes in each predetermined range of values.
 30. A method of investigating particles by their size, comprising the steps of: a. suspending the particles in a liquid; b. moving the particles through a testing passage; c. adjusting the size of said testing passage in accordance with the size of the particles to be measured and to assure an unclogged condition; d. sizing the particles by subjecting to an applied field; and e. triggering one or more actions when the size of a particle is in a range of predetermined values.
 31. An apparatus for classifying particles suspended in a fluid, to provide a suspension of particles, said apparatus comprising: a. a testing passage of a restricted size; b. means for adjustably increasing and decreasing the size of said testing passage in proportion to the size of the particle to be measured, and relieve any existing clogging; c. means for causing the suspension of particles to cross through said testing passage; d. means for providing an applied field within said testing passage; and e. means for detecting the change of the field caused by the passage of each particle through said testing passage.
 32. A method of investigating particles suspended in a liquid which comprises the steps of: a. causing the suspended particles to cross past a predetermined testing volume; b. establishing a steady electric field within the testing volume; c. detecting the change of the field caused by the passage of a particle through the testing volume; and d. separating the desired particles from the particle suspension, according to predetermined values of said change of the field.
 33. An apparatus for classifying particles suspended in a fluid to provide a suspension of particles, said apparatus comprising: a. a testing passage of a restricted size; b. means for adjustably increasing and decreasing the size of said testing passage in proportion to the size of the particle to be detected; c. means for causing the suspension of particles to cross through said testing passage; d. means for providing a magnetic field within said testing passage; e. means for detecting the change of the field caused by the passage of each particle through said testing passage; and f. means for classifying the particles in response to a predetermined range of values of the change of the magnetic field.
 34. A method of investigating particles which comprises the steps of: a. suspending the particles to be investigated in a fluid; b. causing the suspended particles to cross through a testing passage; c. adjusting the size of said testing passage in accordance with the size of the particles to be investigated and to assure an unclogged condition; d. providing a magnetic field within said testing passage; and e. measuring the size of the particles by detecting the change in the field caused by the passage of a particle through said testing passage.
 35. An apparatus for investigating particles suspended in a fluid, to provide a suspension of particles, said apparatus comprising: a. a testing volume restricted as to size according to the size of the particles to be investigated; b. means for adjusting the size of said testing volume; c. means for causing the suspension of particles to cross through said testing volume; d. means for establishing a steady electric field within the testing volume; e. means for measuring the change of the capacitance of said electric field caused by the passage of each particle through said testing volume; f. means for establishing a steady magnetic field within the testing volume; and g. means for measuring the change of the permeability of said magnetic field caused by the passage of each particle through said testing volume.
 36. A combination as defined in claim 35 including means for classifying the particles according to predetermined values of changes of the electric and the magnetic fields.
 37. A method of investigating particles which comprises the steps of: a. suspending the particles to be investigated in a fluid; b. causing the suspended particles to cross past a testing volume; c. changing the size of the testing volume according to the size of the particles to be investigated and to provide an unclogged condition; d. establishing a steady electric field within the testing volume; e. measuring the change of the capacitance of said electRic field caused by the passage of each particle through said testing volume; f. establishing a steady magnetic field within the testing volume; and g. measuring the change of the permeability of said magnetic field caused by the passage of each particle through said testing volume.
 38. An apparatus for studying the dielectric properties of fluids comprising: a. a testing passage; b. means for adjusting in position the size of said testing passage; c. means to cause a sample of the fluid to flow through said testing passage; d. means to produce a steady electric field within said testing passage; e. means to detect the change in capacitance of said field by said fluid; and f. means for diverting a portion of the fluid according to predetermined values of the capacitance change.
 39. Apparatus for investigating particles suspended in a liquid comprising: a. a testing volume; b. means for causing the suspension of particles to cross through said testing volume; c. means for establishing a magnetic field within the testing volume to induce an electrical field in the flowing liquid; d. means, including a pair of pick-up electrodes, for detecting the change of the induced electrical field caused by the passage of each particle through said testing volume and e. means for classifying the particles according to predetermined values of the change detected by said pick-up electrodes.
 40. An apparatus for investigating particles, such as cells, suspended in a transparent liquid, comprising: a. a microscope having a focal plane; b. means, including an opaque diaphragm with an aperture, which coincide with said focal plane, for causing the suspension of cells to flow through said focal plane; c. means for changing the size of the focal plane; d. means for illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane; e. means for detecting the image produced by each particle; f. means, including a light-proof shield, for preventing stray light reaching the image detecting means; g. means for producing an electrical current through said focal plane; and h. means for measuring the variations produced in the electrical current by each particle, as it flows through said focal plane, and thereby determining the size of the particle.
 41. A combination as defined in claim 40 including means for classifying the particles according to predetermined values of the measured characteristics.
 42. A combination as defined in claim 41 including means responsive to a predetermined range of variations of the light caused by at least a portion of the cell, and to a predetermined range of values of the change of the electrical current for actuating said means for classifying said particles.
 43. A combination as defined in claim 41 including means disposed in the vicinity of said testing volume for removing the desired particles from the particle suspension.
 44. A method of investigating particles which comprises the steps of: a. suspending the particles to be investigated in a transparent liquid; b. causing the suspended particles to flow through the focal plane of a microscope, said focal plane having an opaque diaphragm with an aperture; c. changing the size of said aperture in accordance with the size of the particles to be investigated and to assure an unclogged condition; d. illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane; e. detecting the image produced by each particle; f. shielding the image detection against stray-light; g. producing an electrical current through said focal plane; and h. measuring the variations in the electrical current produced by each particle as it flows through said focal plane, and thereby determining the size of the particle.
 45. A method of studying biological cells suspended in a liquid, comprising the steps of: a. measuring the size of the nucleus by the ultraviolet light absorbed by the cell, and at the same time producing an image of the cell; b. measuring the size of the cytoplasm by the disturbance produced in different applied fields by the same cell; c. determining the ratio of nucleus size to cytoplasm size in each cell; and d. multiplying the number of cell images when the nucleus-cytoplasm ratio is within a predetermined range of values, and measuring the amount of light absorbed by a number of areas in which the cell is divided.
 46. An apparatus for investigating particles suspended in a transparent liquid, comprising: a. a microscope having a focal plane; b. means for causing the suspension of particles to flow through said focal plane; c. means for illuminating said focal plane with light absorbed by the particles to thereby produce an image; d. means for measuring the image density, therefore determining the absorption of light produced by a particle; e. means for sizing the particles as they cross said focal plane, thereby triggering the image density-measuring means when the size of a particle is between predetermined values, and f. means responsive to predetermined image characteristics and to a predetermined range of cell-size for removing a cell from the suspension.
 47. An apparatus for investigating particles, such as cells, suspended in a transparent liquid, comprising: a. a microscope having a focal plane; b. means, including an opaque diaphragm with an aperture, which coincide with said focal plane, for causing the suspension of cells to flow through said focal plane; c. means for changing the size of the focal plane; d. means for illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane; e. means for detecting the image produced by each particle; f. means, including a light-proof shield, for preventing stray light reaching the image detecting means; g. means for producing a magnetic field through said focal plane to induce an electrical field in the liquid flowing through said focal plane; and h. means, including a pair of pick-up electrodes, for measuring the variations produced in the electrical field by each particle, as it flows through said focal plane, and thereby determining the size of the particle.
 48. A method of investigating particles which comprises the steps of: a. suspending the particles to be investigated in a transparent liquid; b. causing the suspended particles to flow through the focal plane of a microscope, said focal plane having an opaque diaphragm with an aperture; c. changing the size of said aperture in accordance with the size of the particles to be investigated and to assure an unclogged condition; d. illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane; e. detecting the image produced by each particle; f. shielding the image detection against stray-light; g. producing an electrical field through said focal plane; and h. measuring the variations in the electrical field produced by each particle as it flows through said focal plane, and thereby determining the size of the particle.
 49. An improvement for an apparatus for investigating particles suspended in a liquid, said apparatus having means for producing an electrical current through a testing volume, said improvement comprising: a. means for measuring the variation produced in the electrical current, by each particle, as it flows through the testing volume, and thereby determining its size; b. a microscope having a focal plane which coincides with said testing volume; c. means for changing the size of thE testing volume; d. means for illuminating said focal plane with light that is absorbed by a particle as it flows through said focal plane, and thereby produce an image of the particle; and e. means for detecting the image produced by each particle.
 50. A method of studying biological cells suspended in a liquid comprising the steps of: a. producing an image of a cell through a microscope; b. dividing the image of the cell in a number of areas; c. measuring the light in each one of said areas; d. measuring the size of the nucleus by the number of areas in which the amount of light reaches a predetermined threshold; e. measuring the size of the cytoplasm by the disturbance produced in different applied mediums by the same cell; f. determining the ratio of nucleus size to cytoplasm size; and g. triggering one or more actions when the ratio of nucleus-cytoplasm is within predetermined range of values. 